Method for the production of antiaustrialia antigen antiserum

ABSTRACT

ANTI-AUSTRALIA ANTIGEN ANTISERUM IS PRODUCED BY IMMUNIZNG A WARM-BLOODED ANIMAL WITH A COMPLEX OF AUSTRALIA ANTIGEN AND ANTI-AUSTRALIA ANTIGEN ANTIBODY AND ABOSERBING THE IMMUNE SERUM OF THE IMMUNIZED ANIMAL WITH WATER-INSOLUBILIZED SERUM PROTEIN OF AUSTRALIA ANTIGEN NEGATIVE HUMAN SERUM. THUS-PRODUCED ANTISERUM CONTAINS ANTI-AUSTRALIA ANTIGEN ANTIBODY IN A TITER OF NOT LESS THAN 32 AND GIVES NO POSITIVE REACTION WITH AUSTRALIA ANTIGEN NEGATIVE HUMAN SERUM.

United States Patent 3,830,909 METHOD FOR THE PRODUCTION OF ANTI- AUSTRALIA ANTIGEN ANTISERUM Zenro Hayakawa, Matsumoto, Japan, assignor to Tokyo Standard Serums, Ltd., Tokyo, Japan No Drawing. Filed Nov. 8, 1971, Ser. No. 196,726 Claims priority, applielationsJapan, Nov. 9, 1970,

,9 3 Int. Cl. C12k 1/10; G011] 31/02, 33/16 US. Cl. 424-12 Claims ABSTRACT OF THE DISCLOSURE This invention relates to a method for producing anti- Australia antigen antiserum.

Australia antigen is an antigen which is found at relatively high frequency in the sera of patients with leukaemia and mongolism or the like. This antigen is also found in the sera of 1% or more of normal persons. Since the original studies were done using the serum of an Australian aborigine, this antigen has been called Australia antigen. 70-80% persons among the patients who received transfusion of 200 ml. of blood containing Australia antigen are attacked by serum-haepatitis, the said morbidity rate being far higher than that of persons who received transfusion of normal blood, i.e., not containing the antigen. This fact clearly suggests that Australia antigen is a strong cause of serum-haepatitis after transfusion. Therefore, it is proposed throughout the world to test and eliminate blood containing Australia antigen from all blood pools for transfusion.

The presence of Australia antigen in blood can be checked with the use of an antibody which specifically react with Australia antigen, i.e. anti-Australia antigen antibody, and there have been proposed many test methods such a Micro-Ouchterlony method, Electrosyneresis method, Complement fixation method and Immunoadherence method, all of which employ antiserum containing the said antibody (i.e. anti-Australia antigen antiserum). Thus, enormous amount of anti-Australia antigen antiserum is required for testing all the blood for transfusion. However, it has been diflicult to prepare anti- Australia antigen antiserum of good qualities on an industrial scale. That is, anti-Australia antigen antiserum of human origin has been obtained from the blood of patients with particular diseases e.g. haemophilia and thalassemia who had received transfusions repeatedly. As the production of anti-Australia antigen antiserum of animal origin, there was reported an achievement at a laboratory scale wherein the whole serum of human containing Australia antigen wa injected to rabbits and the resulting immune serum was absorbed with the whole human serum not containing Australia antigen (Nature, Vol. 210, page 1340 et seq. (1966)). However, not only the titer of the objective anti-Australia antigen antibody contained in the immune serum obtained by the said immunization is relatively low but also the immune serum contains many other antibodies caused by the various antigens contained in the whole serum employed for the immunization, and therefore, the antiserum obtained by absorbing this immune serum with the Whole serum contains only a low titer of the objective antibody. Thus, the

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anti-Australia antigen antiserum prepared by the known method can not be utilized for the exact identification and titration of Australia antigen.

Under such technical circumstances, it has been a strong desideratum among the artisans to establish an industrially feasible process for producing anti-Australia antigen antiserum of animal origin which contains a high titer of anti-Australia antigen antibody and which gives a clear reaction selectively with Australia antigen.

The principal object of this invention is to provide a novel and industrially advantageous method for producing anti-Australian antigen antiserum of animal origin.

Another object of this invention is to provide a novel anti-Australia antigen antiserum of animal origin having excellent properties for practical use, i.e. which contains a high titer of anti-Australia antigen antibody and which gives no positive reaction with human serum not containing Australia antigen.

Said objects are realized by immunizing a warmblooded animal with a complex of Australia antigen and anti-Australia antigen antibody and absorbing the im mune serum of the immunized animal with water-insolubilized serum protein of human serum not containing Australia antigen (hereinafter the serum is briefly referred to as Australia antigen negative human serum).

The term warm-blooded animal as used throughout the present specification as well as appended Claims means warm-blooded animal other than human being. As the typical examples of such animals, there may be enumerated guinea pig, rabbit, goat and the like.

The complex of Australia antigen and anti-Australia antigen antibody may be prepared by reacting Australia antigen with anti-Australia antigen antibody in per se established manner for general antigen antibody complexes. Advantageous use is made of complex of Australian antigen and anti-Australia antigen antibody formed as precipitin band in a gel reaction system. Thus, the formation of the complex may be preferably carried out, for example, in the following manner.

A solution of agar in a suitable buffer solution e.g. barbital buffer solution, tri buffer solution or phosphate buffer solution is poured on glass plates so as to give flat agar gel. Two grooves are made in parallel at a suitable interval e.g. 3 to 7 mm. on each agar gel. One of the grooves on each agar gel is charged with a human serum containing Australia antigen (hereinafter briefly referred to as Australia antigen positive human serum), while another of the grooves is charged with anti-Australia antigen antiserum. As the anti-Australia antigen antiserum, there may be employed the antiserum of human origin, the antiserum of warm-blooded animal origin or a mixture thereof.

Then, the agar gels are incubated at room temperature. With the lapse of incubation period, the complex of Australia antigen and anti-Australia antigen antibody is formed as clear single precipitin band between two grooves in each agar gels. Generally, the incubation for about 48 to about 96 hours is sufiicient to form the complex. The said incubation period may be remarkably shortened by applying an electric current to the agar gels in the per se known manner for some Australia antigen testing methods such as Electrosyneresis method. The complex of Australia antigen and anti-Australia antigen antibody thus formed as the precipitin band in the agar gels is recovered from the agar gels. Practically, the precipitin band is recovered by cutting from the Whole agar gel system into a form being adhered to about 1-6 mm. thickness of the gel. Thus-recovered precipitin band is subjected to dialysis against flowing water and subsequently a physiological saline, and then is emulsified. Total dialysis period is advantageously from about 3 to about 8 days.

Thus-treated precipitin band as it is is employable as the complex.

The immunization of the warm-blooded animal With the complex of Australia antigen and anti-Australia antigen antibody is practically effected by injecting the complex into the animal. Especially, intradermal injection gives the good results.

Injection dose, times and intervals of injection may be chosen depending upon the kinds of warm-blooded animals to be immunized and the like. For example, when the above emulsified precipitin band is employed as the complex, advantageous dose at each injection is about 0.1 to 0.5 ml. per guinea pig, about 1 to 5 ml. per rabbit and about 2 to ml. per goat.

Injection is preferably conducted at least twice and repeated till sufficient immunization is attained. The complex may be injected into the animals as the mixture with a suitable adjuvant e.g. Freunds complete or incomplete adjuvant or, with a physiological saline. It is recommended at least for the first injection to employ an emulsion containing Freunds complete adjuvant.

The immune serum is taken from the immunized animals and inactivated by heating in per se established manner.

In the method of this invention, thus-obtained immune serum is subjected to absorption with water-in-solubilized serum protein of Australia antigen negative human serum. The insolubilized serum protein may be prepared by treating Australia antigen negative human serum with an insolubilizing agent by the per .se established method, e.g. described in Immunochemistry, Vol. 6, page 53 et seq. (1969). As the insolubilizing agent, there may be employed, for example, glutaraldehyde, ethyl chloroformate, N-acetylhomocysteine thiolactone, bromoacetyl celulose, p-aminophenylbutyryl-aminoethyl celluose, ethylene-maleic anyhdride copolymer and the like. Among those insolubilizing agents, glutaraldehyde is most advantageously employed. It is recommended to dialyze Australia antigen negative human serum against a physiological saline before treatment with such an insolubilizing agent.

The absorption of the immune serum with the waterinsolubilized serum protein is conducted by homogeneously admixing the immune serum with the Waterinsolubilized serum protein and incubating the resulting mixture at room temperature (about 1 to C.) and then recovering the liquid portion from the mixture. Practically, it is recommended to admix the immune serum with the water-insolubilized serum protein in a volume ratio of from about 1:1 to about 1:5 and incubate the 1 mixture under intermittent shaking. Though incubation period may vary with the above volume ratio and the like, incubation for about 5 to about hours generally gives good results.

Thus-recovered liquid portion can be used, as the anti- Australia antigen antiserum of this invention, per se or diluted with a suitable diluent such as a physiological saline or distilled water, depending on its titer of anti- Australia antigen antibody as Well as the test methods in which the antiserum is employed.

The anti-Australia antigen antiserum thus-prepared is characterized by its high titer of anti-Austrailia antigen antibody as well as by its selective reaction With Australia antigen. That is to say, the anti-Australia antigen antiserum in a form ready for use contains anti-Australia antigen antibody in a titer of not less than 32 and it gives no positive reaction with Australia antigen negative human serum. Throughout the present specification as well as appended Claims, the titer of anti-Australia antigen antibody is determined as the reciprocal number of the highest dilution of the anti-Australia antigen antiserum for exhibiting precipitin band with Australia antigen positive human serum employing Micro-Ouchterlony method as described e.g. in page 964 et seq. of Memorandum on Viral Hepatitis and Tests for the Australia (hepatitisassociated) Antigen and Antibody published by World Health Organization in 1970. In this titration use is made of a standard Australia antigen positive human serum which gives secure positive reactions with anti-Australia antigen antisera of various origins. One of such standard sera is stored under the number S-82728 at the Department of Allergology (Dr. T. Matsuhashi), The Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan.

The anti-Australia antigen antiserum of this invention can be employed for screening the blood for transfusion in optional test methods e.g. described hereinafter similarly to anti-Australia antigen antiserum of human origin.

The test methods employed in Test and Examples are described in the following literatures, respectively.

Electrosyneresis method is described in Igaku No Ayumi (Advances in medicine) published by Ishiyaku Publishers, Inc., Tokyo, Vol. 73, No. 4, page 169 et seq. (1970).

Complement fiixation method is described in Igaku No Ayumi, Vol. 72, No. 12, page 604 et seq. (1970). Immuno-adherence method is described on page 110 et seq. of The Structure and Action of Protein publliggzd by Harper and Row Publisher, New York, in

The following Examples are merely intended to illustrate presently preferred embodiment of this invention and not to restrict the scope of this invention.

The Comparative Example illustrates a typical preparation of the water insolubilized serum protein of Australia antigen negative human serum.

Throughout the foregoing descrption as well as in the following: The Comparative Example, Examples, Test and Claims, mm., mL, M, rpm. and C. respectively refer to millimeter(s), milliliter(s), molar concentration, revolutions per minute and degrees centigrade and percentages are weight/volume unless otherwise specified.

The Comparative Example 100 ml. of Australia antigen negative human serum was dialyzed against a physiological saline at room temperature for 24 hours and then admixed with 10 ml. of 2M-acetate buffer solution of pH 5.0. To the mixture is added dropwise 33 ml. of a 2.5% aqueous solution of glutaraldehyde whereby gel is formed. After the completion of the addition, the mixture is kept standing at room temperature for 5 hours. The gel recovered from the mixture by filtration is washed by centrifugation with 0.01M-phosphate saline buffer of pH 7.4 until the supernatant shows an optical density of not higher than 0.1 at 280 millimicrons. The gel is suspended into 1,000 ml. of a glycine-HCl buffer of pH 2.8 and the suspension is homogenized in a homogenizer. The mixture is centrifuged at 3,000 rpm. for 30 minutes to separate supernatant portion. The resulting fiocculi are suspended into 400 ml. of a mixture of an aqueous 1M-K HPO solution and water in a volume ratio of 1:4, followed by a further homogenization. The mixture is again centrifuged under the same conditions as above and the following fiocculi are Washed by centrifugation with 0.01M-phosphate saline buffer of pH 7.4 until the supernatant shows an optical density of not higher than 0.05 at 280 millimicrons to give the water-insolubilized serum protein which can be employed as the absorbent in the method of this invention.

Example 1 A 1.2% solution of agar in a barbital buffer solution of pH 8.6 was poured on glass plates (25 mm. x 100 mm.) so as to give fiat agar gels of 1.5 mm. thickness. On each agar gel, two grooves (3 mm. in Width x mm. in length x 1.5 mm. in the depth) were made in parallel at an interval of 5 mm. One of the grooves was filled with Australia antigen positive human serum,

6 while another groove was filled with a serum obtained temperature for 12 hours, and then subjected to filtrafrom a haemophiliac who had repeatedly received transtion under reduced pressure with employment of Nutsche fusions, the serum having been confirmed to show an antif n l to i an antiserum, Australia antigen antibody titer of 2. Each of the agar The changes f 5 Serum and 47 serum by the gels was Placed m a humldlfied sealed box and mcubated respective absorption treatments is reactivity against at room temperature for 48 hours, whereby clear single 5 Australia anti en sitive human serum and Australia precipitin band was formed between the two grooves. g P0 The precipitin bands were cut from the gel Plate into antigen negative human serum were determined by Eleca form being adhered to mm thickness of agar gel trosyneresis method and changes in titer of anti-Australia combined each other, dialyzed against flowing water for antigen antibody were determined- T results are 72 hours and washed well with a physiological saline. 10 marized in Tables 1 and 2, respectively.

TABLE 1.-CHANGES IN REACTIVITY Absorbent The whole serum The water-insolubilized serum protein Volume ratio of immune serumzabsorbent Before 1:1 1:2 1:3 1:1 1:2 1:3 absorption Test serum Au(+) Au(-) Au(+) Au(-) Au(+) Au(-) Au(+) Au() Au(+) A11(+) u() 11(+) Au() Immune serum:

R-45 serum 6-17 serum i N orE.-Au(+) =Australia antigen positive human serum; Au() =Australia antigen negative human serum; +=Glear precipitin band formed; =Vague precipitin band termed; -=None precipitin band formed.

The water-insolubi- The whole serum lized serum protein Volume ratio of immune serum:

absorbent 1:1 1:1 Before absorption Test serum----. Au(+) A11(-) Au(+) A1 Immune serum:

R-45 serum o 0 32 0 12s 12s G-17 serum 0 0 64 0 e4 64 No'rE.Au(+)=Australia antigen positive human serum; Au(-) =Australia antigen negative human serum. Half portion of the precipitin band mixture was ho- On the other hand, the above-stored immune sera mogeneously admixed with an equal volume of Freunds 40 other than R- serum and G17 serum were subjected, complete adjuvant to give an emulsion (Emulsion A), respectively, to absorption with an equal volume of the while the remaining portion was homogeneously admixed water-insolubilized serum protein under the same conwith an equal volume of a physiological saline to give ditions as described above to give anti-Australia antigen another emulsion (Emulsion B). antisera.

Emulsion A was intradermally injected to 6 rabbits and 45 Example 2 14 gumea plgs under the 'fonowmg commons: A 0.8% solution of agar in barbital buifer solution of Injection parts Paw and back. pH 8.6 was poured on glass plates (25 x 100 mm.) so as Interval of injection 14 days. to give fiat agar gels of 5 mm. thickness. On each agar Injection times 6. gel, two grooves (3 mm. in width x mm. in length x Dose of one-time injec- Emulsion A prepared from 50 5 mm. in depth) were made in parallel at an interval of tion 4 precipitin bands was 5 mm. One of the grooves was filled with Australia anemployed for one rabbit tigen positive human serum, while another groove was or for 10 guinea pigs. filled with a mixture of anti-Australia antigen antiserum of human origin obtained from a haemophiliac and anti- Australia antigen antiserum of rabbit origin prepared in Example 1 in a volume ratio of about 1:1, the antibody titer of said mixture being 16. Each of the agar gels on the plates was placed in a humidified sealed box and incubated for 72 hours, whereby a clear single precipitin band was formed between the two grooves. The precipitin bands were recovered in a form being adhered to 5 mm. thickness of agar gel from the whole gels by cutting, combined each other, dialyzed against flowing water for 72 hours and subsequently against a physiological saline for 24 hours. The sequence of dialysis was twice repeated. Half portion of the precipitin band mixture was homogeneously admixed with an equal volume of Freunds complete adjuvant to give an emulsion.

The emulsion was intradermally injected to 138 guinea pigs and 10 rabbits at their paws and backs, and to 2 goats At 7 days after the sixth injection, Emulsion B was intradermally injected to the animals at a dose corre sponding that described above. At 10 days after the last injection, immune serum was taken from the immunized animals in per se established manner, inactivated by heating at 56 C. for 30 minutes. The respective sera were 60 stored in a refrigerator after addition of 0.1% of sodium azide.

Among thus-obtained immune sera, the immune serum obtained from an immunized rabbit (designated as R-45 serum) and the immune serum obtained from an immunized guinea pig (designated as 6-17 serum) were subjected to the following absorption treatments:

R-45 serum and 6-17 serum were divided into 6 portions, respectively. Each portion was homogeneously admixed with the water-insolubilized serum portion of 7 Australia antigen negative human serum prepared in at their back and bell at the doses f 0 5 m1 r The Comparative Example or the whole serum of pig, 5 1 per rabbit i 10 1 goat Pe gumea A allilgen llegatlve human Serum 111 ihe V111me At 49 days after the said injection, another emulsion ratios listed below m Table 1. Each of the resulting mixprepared by admixing the remaining portion of the p tures was incubated under intermittent shaking at room cipitin band mixture with an equal volume of a physiological saline was intradermally injected to the respective animals at the same doses as described above.

At days after the last injection, immune serum was taken from each immunized animal in per se established manner, and inactivated by heating at 56 C. for 30 minutes. After addition of 0.1% of sodium azide, the respective sera were stored in a refrigerator.

The immune sera were subjected, respectively, to the absorption with an equal volume of the WHEY-11150111131 lized serum protein of Australia antigen negative human serum prepared in Reference under the same conditions as described in Example 1 to give anti-Australia antigen antisera.

The titer of anti-Australia antigen antibody was determined With regard to the respective antisera.

The mean antibody titers for the guinea pigs antisera, rabbits antisera and goats antisera are listed in Table 3.

, TABLE 3 Antisera Mean antibody titer Of guinea pig origin 128 Of rabbit origin 64 Of goat origin 32 Test Sera of forty human subjects were tested employing anti-Australia antigen antiserum of rabbit origin and anti- 8 by the reaction between Australia antigen positive human serum and anti-Australia antigen antiserum in an agar gel, and absorbing the immune serum of the immunized animal with water-insolubilized serum protein of Australia antigen negative human serum.

2. The method of claim 1, wherein the warm-blooded animal is selected from the group consisting of guinea pig, rabbit and goat.

3. The method of claim 1, wherein the immunization is carried out by intradermally injecting the complex into the warm-blooded animal.

4. The method of claim 1, wherein the absorption is carried out by homogenously admixing the immune serum with the water-insolubilized serum protein in :1 volume ratio of from about 1:1 to about 1:5, incubating the resulting mixture at room temperature for about 5 to about hours and then recovering the liquid portion from the mixture.

5. The method of claim 1, wherein the water-insolubilized serum protein is prepared by treating Australia antigen negative human serum with glutaraldehyde.

TABLE 4 Test method Micro- Electro- Complement Quehterlony syneresis Immune-adherence fixation method method method method Antiserilm and itsdilution G, H, R, o, H, R. G, faetorintest 1:1 1:1 1:1 1:1 1:16 1:1 1:20 1:20 1.15 1:32 1:8 1:1

Subject number:

7 +(2 +(2 +(2 i2 $12 I I I +(2 2- i i I Determination was impossible.

Nor1-:.-R=Anti-Anstralia antigen antiserum of rabbit origin; G =Anti-Austra1ia antigen antiserum of guinea pig origin; H=Anti-Aus tral1a antigen antiserum o1 human origin, +=Positive reaction; i=Vague positive reaction; =No positive reaction.

Australia antigen antiserum of guinea pig origin, both having been prepared in Example 1, and anti-Australia antigen antiserum of human origin obtained from a haemophiliac who had repeatedly received transfusions, by Micro-Ouchterlony method, Electrosyneresis method, Immune-adherence method and Complement fixation method.

The results are summarized in Table 4, wherein the data obtained on the sera of thirty-three subjects which indicated no positive reaction against all the antisera are omitted.

The said results clearly demonstrate that anti-Australia antigen antiserum of animal origin of the present inven tion gives specific and clear reaction with Australia antigen positive human sera, and therefore, that it can be employed for screening the blood for transfusion similarly to anti-Australia antigen antiserum of human origin.

Having thus disclosed the invention, what is claimed is:

1. A method for producing anti-Australia antigen antiserum which comprises immunizing a warm-blooded animal with a complex which is the precipitin band formed References Cited UNITED STATES PATENTS ALBERT T. MEYERS, Primary Examiner A. P. FAGELSON, Assistant Examiner US. Cl. X.R. 

